Composition with preventive or improvement effect on symptoms or diseases associated with stress-induced behavior disorders

ABSTRACT

A composition with a preventive or improvement effect on symptoms or diseases associated with stress-induced behavior disorders, comprising arachidonic acid and/or a compound comprising arachidonic acid as a constituent fatty acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Application No. 11/663,076filed Mar. 16, 2007, which is the National Stage of InternationalApplication No, PCT/P2005/005623 filed Mar. 18, 2005, which claimsbenefit of Japanese Patent Application No. 2004-271927, filed on Sep.17, 2004, and which are incorporated by reference herein in theirentirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a preventive or improvement agent forsymptoms or diseases associated with stress-induced behavior disorders,comprising as an active ingredient arachidonic acid and/or a compoundcomprising arachidonic acid as a constituent fatty acid, as well as to acomposition with a preventive or improvement effect on symptoms ordiseases associated with stress-induced behavior disorders, and a methodfor its production. More specifically, the invention relates to apreventive or improvement agent for stress-induced habits such as fingersucking and onychophagy, adjustment disorder, attention deficithyperactivity disorder, acute stress disorder or posttraumatic stressdisorder, comprising as an active ingredient at least one selected fromthe group consisting of arachidonic arachidonic acid alcohol esters, andtriglycerides, phospholipids or glycolipids wherein all or a portion ofthe constituent fatty acid is arachidonic acid, as well as to a food orbeverage with such a preventive or improvement effect and a method forits production.

BACKGROUND ART

Stress is recognized as a response which can lead to behavior disorders,and the symptoms associated with stress-induced behavior disorders areknown as stress-related disorders; these are classified into severaltypes of symptoms based on two factors: the nature of the stress and thedisposition of the individual experiencing the stress. The symptoms spana wide range from simple habits such as finger sucking or onychophagyseen primarily during the infant period and resulting from light stressfactors, to adjustment disorder and attention deficit hyperactivitydisorder which are considered to be highly influenced by individualdisposition, and further to acute stress disorder or posttraumaticstress disorder, which are associated with extremely intense stressbeyond individual disposition (Shindan to Chiryo 91, 1333, 2003).

Recently, increased blood IL-1β has been reported in post-traumaticstress disorder patients (Biol. Psychiatry 42, 345, 1997), and researchhas focused on the relationship between IL-1β and neuronopathy.

Drugs used for stress-related disorders include benzodiazepine-baseddrugs used for insomnia and anxiety, serotonin reuptake inhibitors andtricyclic antidepressant drugs used to alleviate symptoms flashbacks,adrenaline antagonists and anticonvulsant drugs used for symptoms ofhypervigilance, and antipsychotic agents used for exaggerated startleresponse or increased irritability. However, all such agents aresymptomatic treatment for improvement of superficial symptoms such asdepression, insomnia and excitement, and unfortunately no agents forcausal treatment exist at this time.

One possible treatment for stress-related disorders is to suppress theirprogression by removing the cause of stress. However, given the modernenvironment it is very difficult to eliminate the causative factors ofstress.

Thus, to date no drug has existed which is effective as a pharmaceuticalagent having a preventive or improvement effect on symptoms or diseasesassociated with stress-induced behavior disorders. Furthermore,applications to food products have been hampered by the limitation tocomponents which produce no side effects.

The brain consists of a lipid mass-like tissue, with phospholipidsconstituting about ⅓ of the white matter and about ¼ of the gray matter.The polyunsaturated fatty acids in phospholipids of the various cellmembranes in the brain consist primarily of arachidonic acid anddocosahexaenoic acid. However, arachidonic acid and docosahexaenoic acid(DHA) cannot be synthesized de novo in animal bodies and must bedirectly or indirectly obtained through diet (for example, as thearachidonic acid and docosahexaenoic acid precursors, linoleic acid andα-linolenic acid).

Burgess et al. have demonstrated that arachidonic acid and DHA contentsof plasma phospholipids are significantly lower in posttraumatic stressdisorder patients (Am J Clin Nutr 71, 327S, 2000). It has also beenreported that liver microsome Δ5-desaturase and Δ6-desaturase activityis reduced in separately bred stress model rats (Proc Soc Exp Biol Med.205, 56, 1994), and the reduction in activity of these desaturases isbelieved to be responsible for a lack of brain levels of arachidonicacid and DHA, polyunsaturated fatty acids with a high degree ofunsaturation.

On the other hand, several experiments have been reported usingadministration of arachidonic acid to animal stress models. Song et al.reported that administration of free arachidonic acid was not effectivefor IL-1β induced stress anxiety behavior models (J Lipid Res. 44, 1984,2003). Also, Clements et al. describe giving attention deficithyperactivity disorder (ADHD) models (spontaneously hypertensive rats,SHR) feed containing 0.5% arachidonic acid and 0.9% DHA for 8 weeks, andreported increased DHA in the brain phospholipids but no observableeffect (Dev Psychobiol. 43, 57, 2003). No other reports indicate thatadministration of arachidonic acid improves stress-induced behaviordisorders.

Thus, while it has been reported that arachidonic acid levels in thebody are lowered by stress, it has not been clearly demonstrated whetherarachidonic acid or compounds including arachidonic acid as aconstituent fatty acid according to the invention are effective for theprevention or improvement of symptoms or diseases associated withstress-induced behavior disorders, and in fact the experiments conductedto date have been definitively negative.

-   Non-patent document 1: Shindan to Chiryo 91, 1333, 2003-   Non-patent document 2: Biol. Psychiatry 42, 345, 1997-   Non-patent document 3: Am J Olin Nutr 71, 327S, 2000-   Non-patent document 4: Proc Soc Exp Biol Med. 205, 56, 1994-   Non-patent document 5: J Lipid Res. 44, 1984, 2003-   Non-patent document 6: Dev Psychobiol. 43, 57, 2003

DISCLOSURE OF THE INVENTION

Thus, a strong demand exists for development of pharmaceuticals whichprevent and improve symptoms or diseases associated with stress-inducedbehavior disorders, as well as such compounds which are highly suitablefor consumption and lacking notable side effects.

As a result of much diligent research conducted with the purpose ofelucidating the preventive or improvement effects on symptoms ordiseases associated with stress-induced behavior disorders by agentscomprising as active ingredients arachidonic acid and/or compoundscomprising arachidonic acid as a constituent fatty acid, the presentinventors found that the active ingredients of the invention exhibitapparent behavioral pharmacologic effects in a behavioral observationtest using mice subjected to restraint stress, which is considered toapproximate human emotional stress.

We also succeeded in realizing industrial production of a triglyceridecontaining at least 10% microbially generated arachidonic acid, andsupplied the triglyceride for testing in order to elucidate the effectof the invention.

Specifically, the present invention provides a preventive or improvementagent for symptoms or diseases associated with stress-induced behaviordisorders and a composition with a preventive or improvement effect onsymptoms or diseases associated with stress-induced behavior disorders,comprising as an active ingredient arachidonic acid and/or a compoundcomprising arachidonic acid as a constituent fatty acid, as well as amethod for their production. More specifically, the invention provides apreventive or improvement agent for stress-induced habits such as fingersucking and fingernail biting, adjustment disorder, attention deficithyperactivity disorder, acute stress disorder or posttraumatic stressdisorder, comprising as an active ingredient at least one selected fromthe group consisting of arachidonic acid, arachidonic acid alcoholesters, and triglycerides, phospholipids or glycolipids wherein all or aportion of the constituent fatty acid is arachidonic acid, as well as toa composition with such a preventive or improvement effect and a methodfor its production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results for Example 3, indicating theeffect of arachidonic acid on the rearing behavior of stressed mice.

FIG. 2 is a graph showing the results for Example 3, indicating theeffect of arachidonic acid on the ultromotivity of stressed mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a preventive or improvement agent forsymptoms or diseases associated with stress-induced behavior disordersand a composition with a preventive or improvement effect on symptoms ordiseases associated with stress-induced behavior disorders, comprisingas an active ingredient arachidonic acid and/or a compound comprisingarachidonic acid as a constituent fatty acid, as well as a method fortheir production.

As “symptoms or diseases associated with stress-induced behaviordisorders” there may be mentioned habits such as finger sucking andonychophagy, adjustment disorder, attention deficit hyperactivitydisorder, acute stress disorder or posttraumatic stress disorder, butthe symptoms and diseases are not limited to these and include allsymptoms and diseases associated with stress-induced behavior disorders.

The active ingredient of the invention is arachidonic acid, but anycompound comprising arachidonic acid as a constituent fatty acid may beused. As compounds comprising arachidonic acid as a constituent fattyacid there may be mentioned arachidonic acid salts, such as calcium orsodium salts. There may also be mentioned arachidonic acid lower alcoholesters such as arachidonic acid methyl ester and arachidonic acid ethylester. There may also be used triglycerides, phospholipids orglycolipids wherein all or a portion of the constituent fatty acid isarachidonic acid. However, the invention is not limited to the compoundsmentioned above, and includes any compound comprising arachidonic acidas a constituent fatty acid.

For application to food products, the arachidonic acid is preferably inthe form of a triglyceride or phospholipid, and most preferably in theform of a triglyceride. While virtually no natural sources ofarachidonic acid-containing triglycerides (i.e., triglycerides includinga triglyceride wherein all or a portion of the constituent fatty acid isarachidonic acid) exist, the present inventors have been the first toclearly demonstrate that it is possible to industrially utilizetriglycerides comprising arachidonic acid as a constituent fatty acid,that the active ingredients of the invention exhibit apparent behavioralpharmacologic effects in mice subjected to restraint stress andevaluated by a behavioral observation test and have preventive orimprovement effects for symptoms or diseases associated withstress-induced behavior disorders, and that the effects are attributableto arachidonic acid.

According to the invention, therefore, triglycerides including atriglyceride wherein all or a portion of the constituent fatty acid isarachidonic acid (arachidonic acid-containing triglycerides) may be usedas the active ingredients of the invention. For application in foods,the arachidonic acid-containing triglycerides are preferably oils orfats (triglycerides) in a form wherein the arachidonic acid content ofthe total constituent fatty acid of the triglycerides is at least 10 wt% (w/w), more preferably at least 20 wt %, even more preferably at least30 wt %, and most preferably at least 40 wt %. Thus, the presentinvention may employ any such compounds which are obtained by culturingmicroorganisms capable of producing arachidonic acid-containing oils orfats (triglycerides).

As microorganisms capable of producing oils or fats (triglycerides)containing arachidonic acid; there may be mentioned microorganismsbelonging to the genera Mortierella, Conidiobolus, Pythium,Phytophthora, Penicillium, Cladoporium, Mucor, Fusarium, Aspergillus,Rhodotorula, Entornophthora, Echinosporangium and Sapro egnia.

As examples of microorganisms belonging to the genus Mortierella,subgenus Mortierella, there may be mentioned Mortierella elongate,Mortierella exigua, Mortierella hygrophila and Mortierella alpina. Morespecifically, there may be mentioned the strains Mortierella elongateIFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, andMortierella alpine IFO8568, ATCC16266, ATCC32221, ATCC42430, CBS219.35,CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70,CBS754.68, etc.

All of these strains may be acquired without any special restrictionsfrom the Institute for Fermentation, Osaka (IFO), American Type CultureCollection (ATCC) or Centralbureau voor Schimmelcultures (CBS). Theremay also be used the strain Mortierella elongata SAM0219 (FERM-P 8703)(deposited under the provisions of the Budapest Treaty on Mar. 19, 1986with the Patent Microorganism Depository of National Institute ofIndustrial Science and Technology at Chuo 6, 1-1, Higashi 1-chome,Tsukuba city, Ibaraki pref., Japan, as FERM BP-1239), isolated from soilby the research group for the present invention.

For culturing of a strain to be used for the invention, spores, hypha ora pre-culture solution obtained by pre-culturing the strain may beseeded in a liquid medium or solid medium for culturing. In the case ofliquid culturing, the carbon source used may be a common one such asglucose, fructose, xylose, saccharose, maltose, soluble starch,molasses, glycerol or mannitol, although there is no limitation tothese.

As nitrogen sources there may be used organic nitrogen sources includingurea, and natural nitrogen sources such as peptone, yeast extract, maltextract, meat extract, casamino acid, corn, steep liquor, soybeanprotein, defatted soybean and cotton seed meal, or inorganic nitrogensources such as sodium nitrate, ammonium nitrate and ammonium sulfate.Trace nutrient sources including inorganic salts such as phosphoric acidsalts, magnesium sulfate, iron sulfate and copper sulfate, or vitamins,may also be used if necessary. The medium components are notparticularly restricted so long as they are in concentrations which donot prevent growth of the microorganisms. For most practicalapplications the carbon source may be used at a concentration of 0.1-40wt % and preferably 1-25 wt %. The initial nitrogen source addition maybe at 0.1-10 wt % and preferably 0.1-6 wt %, with further feeding of thenitrogen source during culturing.

By controlling the carbon source concentration of the medium it ispossible to obtain oils or fats (triglyceride) containing at least 45 wt% arachidonic acid as the active ingredient of the invention. The cellgrowth phase is the culturing period up to the 2nd-4th day of culturing,while the fat/oil accumulation phase is from the 2nd-4th day ofculturing. The initial carbon source concentration is 1-8 wt % andpreferably 1-4 wt %, with successive supplemental addition of the carbonsource only between the cell growth phase and the early fat/oilaccumulation phase, for a total supplemental carbon source addition of2-20 wt % and preferably 5-15 wt %. The amount of carbon source addedbetween the cell growth phase and the early fat/oil accumulation phasewill depend on the initial nitrogen source concentration, and if thecarbon source concentration in the medium is 0 from the 7th day ofculturing, preferably from the 6th day of culturing and more preferablyfrom the 4th day of culturing, it will be possible to obtain oils orfats (triglyceride) containing at least 45 wt % arachidonic acid, as theactive ingredient of the invention.

The culturing temperature for the arachidonic acid-producing cells willdiffer depending on the microorganism used, but is 5-40° C., preferably20-30° C., while culturing at 20-30° C. for proliferation of the cellsmay also be followed by continued culturing at 5-20° C. to produceunsaturated fatty acids. Such temperature control can also be utilizedto increase the proportion of polyunsaturated fatty acids among theproduced fatty acids. The pH of the medium may be 47-10 and preferably5-9, for jar fermentor culturing, shake culturing or stationaryculturing. The culturing is normally carried out for 2-30 days,preferably 5-20 days and more preferably 5-15 days.

In addition to controlling the carbon source concentration of the mediumas a strategy for increasing the proportion of arachidonic acid in thearachidonic acid-containing oils or fats (triglyceride), arachidonicacid-rich oils or fats may also be obtained by selective hydrolysis ofthe arachidonic acid-containing oils or fats. Since lipases used forsuch selective hydrolysis do not have regiospecificity for triglyceridesand the hydrolytic activity decreases in proportion to the number ofdouble bonds, the ester bonds of the fatty acids other than thepolyunsaturated fatty acids are preferentially hydrolyzed. Furthermore,ester-exchange reaction between the produced PUFA glycerides may be usedto produce triglycerides with an increased polyunsaturated fatty acidcontent (“Enhancement of Arachidonic Acid: Selective Hydrolysis of aSingle-Cell Oil from Mortierella with Candida cylindracea Lipase”: J.Am. Oil Chem. Soc., 72, 1323-1327, 1998).

Thus, oils or fats (triglyceride) with a high content of arachidonicacid obtained by selective hydrolysis of arachidonic acid-containingoils or fats can be prepared as the active ingredient of the invention.The proportion of arachidonic acid with respect to the total fatty acidcontent of the arachidonic acid-containing oils or fats (triglyceride)of the invention is preferably higher from the standpoint of eliminatingthe effect of other fatty acids, but it does not necessarily have to bea high proportion, and in fact the absolute amount of arachidonic acidcan pose a problem for application to some foods. Oils or fats(triglycerides) containing arachidonic acid at 10 wt % or greater can besuitably used in most cases.

As triglycerides wherein all or a portion of the constituent fatty acidis arachidonic acid according to the invention, there may be usedtriglycerides having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position. The oils orfats (triglycerides) used may also comprise at least 5 mole percent,preferably at least 10 mole percent, more preferably at least 20 molepercent and most preferably at least 30 mole percent, of triglycerideshaving medium chain fatty acids bonded at the 1,3-positions andarachidonic acid bonded at the 2-position. The medium chain fatty acidsbonded at the 1,3-positions of the triglyceride may be selected fromamong C6-12 fatty acids. As examples of C6-12 fatty acids there may bementioned caprylic acid or capric acid, with1,3-capryloyl-2-arachidonoyl-glycerol (hereinafter, “8A8”) beingparticularly preferred.

Such triglycerides having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position are optimumoils or fats (triglycerides) for elderly persons. Generally speaking,ingested oils or fats (triglycerides) are hydrolyzed by pancreaticlipases upon entering the small intestine, but since pancreatic lipasesare 1,3-specific, the 1,3-positions of the triglycerides are cleaved toform two free fatty acids while simultaneously producing a single2-monoacylglycerol (MG). As 2-MG has extremely high bile solubility andis highly absorbable, the 2-position fatty acid is generally consideredto be better absorbed. In addition, 2-MG dissolved in bile acid acts asa surfactant and thus increases the absorption of the free fatty acids.

The free fatty acids and 2-MG then form bile acid complex micellestogether with cholesterol, phospholipids and the like and areincorporated into the intestinal epithelial cells where triacylglycerolsare resynthesized, being finally released into the lymph aschylomicrons. However, the fatty acid specificity of pancreatic lipasesis higher for saturated fatty acids, whereas arachidonic acid is not aseasily cleaved. Another problem is that pancreatic lipase activitydeclines with age, and therefore triglycerides having medium chain fattyacids bonded at the 1,3-positions and arachidonic acid bonded at the2-position are more optimal oils or fats (triglycerides) for theelderly.

One specific production method for triglycerides having medium chainfatty acids bonded at the 1,3-positions and arachidonic acid bonded atthe 2-position is a method using a lipase which acts only on the1,3-position ester bonds of triglycerides, in the presence ofarachidonic acid-containing oils or fats (triglyceride) and a mediumchain fatty acid.

The oils or fats (triglyceride) starting material are a triglyceridecomprising arachidonic acid as a constituent fatty acid, but in the caseof a high proportion of arachidonic acid with respect to the totalconstituent fatty acid of the triglycerides, reduced reaction yield dueto excess unreacted oils or fats (the triglyceride starting material andtriglycerides wherein only one of the 1,3-position-fatty acids has beenconverted to a medium chain fatty acid) can be prevented if thetemperature is above the normal enzyme reaction temperature of 20-30°C., such as 30-50° C. and preferably 40-50° C.

As examples of lipases which act specifically on the 1,3-position esterbonds of triglycerides there may be mentioned lipases produced bymicroorganisms such as Rhizopus, Rhizomucor and Aspergillus, as well asporcine pancreatic lipases. Any such commercially available lipases maybe used. For example, there may be mentioned Rhizopus delemar lipase(Talipase, Tanabe Pharmaceutical Co., Ltd.), Rhizomucor miehei lipase(Ribozyme IM, Novo Nordisk Co., Ltd.) and Aspergillus niger lipase(Lipase A, Amano Pharmaceutical Co., Ltd.), although there is nolimitation to these enzymes and any 1,3-specific lipases may be used.

The form of the lipase used is preferably an immobilized form on animmobilizing support in order to impart heat resistance to the enzyme,since the reaction temperature is 30° C. or above and preferably 40° C.or above for increased reaction efficiency. The immobilizing support maybe a porous (highly porous) resin, for example, an ion-exchange resinwith pores of approximately 100 Å or greater such as Dowex MARATHON WBA.However, this condition is not restrictive on the immobilizing support,and any immobilizing support capable of imparting heat resistance may beused.

The immobilizing support may be suspended in an aqueous solution of a1,3-specific lipase at a weight proportion of 0.5-20 of the latter withrespect to the former, and a 2- to 5-fold amount of cold acetone (forexample, −80° C.) may be slowly added to the suspension while stirringto form a precipitate. The precipitate may then be dried under reducedpressure to prepare the immobilized enzyme. As a simpler method, a1,3-specific lipase in a proportion of 0.05-0.4 with respect to theimmobilizing support may be dissolved in a minimal amount of water andmixed with the immobilizing support while stirring and dried underreduced pressure to prepare the immobilized enzyme. This procedure canimmobilize approximately 90% lipase on the support, but since absolutelyno ester exchange activity will be exhibited in that state, pretreatmentmay be carried out in a substrate containing 1-10 wt % (w/v) water andpreferably a substrate containing 1-3 wt % water, in order to activatethe immobilized enzyme to maximum efficiency before it is provided forproduction.

The amount of water added to the reaction system is extremely importantdepending on the type of enzyme, because a lack of water will impedeester exchange while an excess of water will cause hydrolysis and areduced glyceride yield (since hydrolysis will produce diglycerides andmonoglycerides). However, if the immobilized enzyme used has beenactivated by pretreatment the amount of water added to the reactionsystem is no longer crucial, and an efficient ester exchange reactioncan be carried out even in a completely water-free system. Also,selection of the type of enzyme agent may allow the pretreatment step tobe omitted.

Thus, by using a heat-resistant immobilized enzyme and raising theenzyme reaction temperature, it is possible to efficiently producetriglycerides having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position (8A8),without lowering the reaction efficiency even for arachidonicacid-containing oils or fats (triglycerides) with low reactivity for1,3-specific lipases.

A method for production of a dietary product having a preventive orimprovement effect on symptoms or diseases associated withstress-induced behavior disorders, involves adding arachidonic acidand/or a compound including arachidonic acid as a constituent fatty acidalone, or in combination with a dietary material containingsubstantially no arachidonic acid or only a slight amount thereof. Here,a “slight amount” means that even if arachidonic acid is present in thedietary product material and a food composition containing it isingested by a human, the amount does not reach the daily amount ofarachidonic acid consumption according to the invention, as describedhereunder.

An unlimited number of uses exist for oils or fats (triglycerides)wherein all or a portion of the constituent fatty acid is arachidonicacid: for example, they may be used as starting materials and additivesfor foods, beverages, cosmetics and pharmaceuticals. The purposes of useand amounts of use are also completely unrestricted.

As examples of food compositions there may be mentioned ordinary foods,as well as functional foods, nutritional supplements, food for specifiedhealth uses, preterm infant formula, term infant formula, infant foods,maternal foods or geriatric foods. As examples of fat/oil-containingfoods there may be mentioned natural fat/oil-containing foods such asmeat, fish and nuts, foods to which oils or fats are added duringpreparation, such as soups, foods employing oils or fats as heatingmedia, such as donuts, oils or fats foods such as butter, processedfoods to which oils or fats are added during processing, such ascookies, or foods which are sprayed or coated with oils or fats uponfinishing, such as hard biscuits. Such compositions may also be added toagricultural foods, fermented foods, livestock feeds, marine foods andbeverages which contain no oils or fats. They may also be in the form offunctional foods or pharmaceuticals, and for example, in processed formsuch as enteral nutrients, powders, granules, lozenges, oral solutions,suspensions, emulsions, syrups and the like.

A composition of the invention may also contain various carriers oradditives ordinarily used in foods and beverages, pharmaceuticals orquasi drugs, in addition to the active ingredient of the invention.Antioxidants are particularly preferred as additives to preventoxidation of the active ingredient of the invention. As examples ofantioxidants there may be mentioned natural antioxidants such astocopherols, flavone derivatives, phyllodulcins, kojic acid, gallic acidderivatives, catechins, fukiic acid, gcssypol, pyrazine derivatives,sesamol, guaiaol, guaiac acid, p-coumaric acid, nordihydroguaiareticacid, sterols, terpenes, nucleotide bases, carotenoids, lignans and thelike, and synthetic antioxidants including ascorbic palmitic acidesters, ascorbic stearic acid esters, butylhydroxyanisole (BHA),butylhydroxytoluene (BHT), mono-t-butylhydroquinone (TBHQ) and4-hydroxymethyl-2,6-di-t-butylphenol (HMBP).

As tocopherols there may be mentioned α-tocopherol, β-tocopherol,γ-tocopherol, δ-tocopherol, ε-tocopherol, ζ-tocopherol, η-tocopherol andtocopherol esters (tocopherol acetate and the like), as well astocopherol analogs. As examples of carotenoids there may be mentionedβ-carotene, cantaxanthine, astaxanthine and the like.

The composition of the invention may also contain, in addition to theactive ingredient of the invention, supports such as carrier supports,extenders, diluents, bulking agents, dispersing agents, excipients,binder solvents (for example, water, ethanol and vegetable oils),dissolving aids, buffering agents, dissolving accelerators, gellingagents, suspending agents, wheat flour, rice flour, starch, corn starch,polysaccharides, milk protein, collagen, rice oil, lecithin and thelike. As examples of additives there may be mentioned vitamins,sweeteners, organic acids, coloring agents, aromatic agents,moisture-preventing agents, fibers, electrolytes, minerals, nutrients,antioxidants, preservatives, fragrances, humectants, natural foodextracts, vegetable extracts and the like, although there is nolimitation to these.

Arachidonic acid is the main active ingredient of the compound which iseither arachidonic acid or comprises arachidonic acid as a constituentfatty acid. The daily intake of arachidonic acid from dietary sourceshas been reported to be 0.14 g in the Kanto region and 0.19-0.20 g inthe Kansai region of Japan (Shishitsu Eiyougaku 4, 73, 1995), and inconsideration of reduced oils or fats intake and reduced pancreaticlipase function in the elderly, a correspondingly greater amount ofarachidonic acid must be ingested. Thus, the daily intake of thearachidonic acid or the compound comprising arachidonic acid as aconstituent fatty acid according to the invention for an adult (forexample, 60 kg body weight) is 0.001-20 g, preferably 0.01-10 g, morepreferably 0.05-5 g and most preferably 0.1-2 g, based on thearachidonic acid content.

When the active ingredient of the invention is to be actually appliedfor a food or beverage product, the absolute amount of arachidonic acidin the product is an important factor. However, since the absoluteamount added to foods and beverages will differ depending on the amountof consumption of those foods or beverages, triglycerides including atriglyceride wherein all or a portion of the constituent fatty acid isarachidonic acid may be added to food products in amounts of at least0.001 wt %, preferably at least 0.01 wt % and more preferably at least0.1 wt % in terms of arachidonic acid. For addition to food and beverageproducts of triglycerides having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position, the amountmay be at least 0.0003 wt %, preferably at least 0.003 wt % and morepreferably at least 0.03 wt %.

When the composition of the invention is to be used as a pharmaceutical,it may be produced according to a common method in the field ofpharmaceutical preparation techniques, such as according to a methoddescribed in the Japanese Pharmacopeia or a similar method.

When the composition of the invention is to be used as a pharmaceutical,the content of the active ingredient in the composition is notparticularly restricted so long as the object of the invention isachieved, and any appropriate content may be employed.

When the composition of the invention is to be used as a pharmaceutical,it is preferably administered in the form of an administrable unit, andespecially in oral form. The dosage of the composition of the inventionwill differ depending on age, body weight, symptoms and frequency ofadministration, but for example, the arachidonic acid and/or compoundincluding arachidonic acid as a constituent fatty acid according to theinvention may be administered at about 0.001-20 g, preferably 0.01-10 g,more preferably 0.05-5 g and most preferably 0.1-2 g (as arachidonicacid) per day for adults (approximately 60 kg), either once a day ordivided among multiple doses, such as three separate doses.

The major fatty acid components of phospholipid membranes in the brainare arachidonic acid and docosahexaenoic acid, and therefore from thestandpoint of balance, a combination with docosahexaenoic acid ispreferred. Also, since the proportion of eicosapentaenoic acid in brainphospholipid membranes is very small, a combination of arachidonic acidand docosahexaenoic acid containing virtually no eicosapentaenoic acidis especially preferred. Furthermore, the arachidonicacid/docosahexaenoic acid ratio in the combination of the arachidonicacid and docosahexaenoic acid is preferably in the range of 0.1-15, andmore preferably in the range of 0.25-10. Also, the amount ofeicosapentaenoic acid in the food or beverage preferably does not exceed⅕ of the arachidonic acid (weight ratio).

EXAMPLES

The present invention will now be explained in greater detail by thefollowing examples, with the understanding that the invention is notlimited to these examples.

Example 1 Method for Production of Arachidonic Acid-ContainingTriglycerides

Mortierella alpina CBS754.68 was used as the arachidonic acid-producingstrain. After preparing 6 kL of medium containing 1.8% glucose, 3.1%defatted soybean powder, 0.1% soybean oil, 0.3% KH₂PO₄, 0.1% Na₂SO₄,0.05% CaCl₂·2H₂O and 0.05% MgCl₂·6H₂O in a 10 kL culturing tank, theinitial pH was adjusted to 6.0.

A 30 L portion of the preculturing solution was transferred for 8 daysof jar fermentor culturing under conditions with a temperature of 26°C., an airflow of 360 m³/h and an internal pressure of 200 kPa. Thestirring rate was adjusted to maintain a dissolved oxygen concentrationof 10-15 ppm. Also, the glucose concentration was adjusted by thefeeding culture method for a glucose concentration in the range of1-2.5% in the medium up to the 4th day, with 0.5-1% maintainedthereafter (where the percentage values are weight (W/V)%).

After completion of the culturing, the cells containing triglycerideshaving arachidonic acid as constituent fatty acid were collected byfiltration and drying, and the fat and oil portion was extracted fromthe collected cells by hexane extraction and subjected to dietary oilsor fats purification steps (degumming, deoxidation, deodorization,decolorizing) to obtain 150 kg of arachidonic acid-containingtriglycerides (triglycerides including a triglyceride wherein all or aportion of the constituent fatty acid is arachidonic acid). The obtainedoils or fats (triglycerides) were methylesterified, and the obtainedfatty acid methyl ester mixture was analyzed by gas chromatography andfound to have an arachidonic acid proportion of 40.64 wt % of the totalfatty acid.

The contents of palmitic acid, stearic acid, oleic acid, linoleic acid,γ-linolenic acid and dihomo-γ-linolenic acid were 11.63%, 7.45%, 7.73%,9.14%, 2.23% and 3.27% by weight, respectively. The arachidonicacid-containing oils or fats (triglycerides) (TGA40S) were alsoethylesterified, and the fatty acid ethyl ester mixture including 40 wt% arachidonic acid ethyl ester was separated and purified by anestablished high-performance liquid chromatography method to obtain 99wt % arachidonic acid ethyl ester.

Example 2 Production of triglycerides including at least 5 mole percent8A8

After suspending 100 g of an ion-exchange resin carrier (Dowex MARATHONWBA: Dow Chemical) in 80 ml of Rhizopus delemar lipase aqueous solution(12.5% Talipase powder, Tanabe Pharmaceutical Co., Ltd.), 240 ml of coldacetone (−80° C.) was stirred therewith and the mixture was dried underreduced pressure to obtain the immobilized lipase.

Next, 80 g of the triglycerides containing 40 wt % arachidonic acid(TGA40S) obtained in Example 1, 160 g of caprylic acid, 12 g of theaforementioned immobilized lipase and 4.8 ml of water were reacted for48 hours at 30° C. while stirring (130 rpm). Upon completion of thereaction, the reaction solution was removed to obtain the activatedimmobilized enzyme.

A 10 g portion of immobilized lipase (Rhizopus delemar lipase, carrier:Dowex MARATHON WBA) was than packed into a jacketed glass column(1.8×12.5 cm, 31.8 ml volume), and the reaction oils or fats comprisinga mixture of the TGA40S obtained in Example 1 and caprylic acid(TGA40S:caprylic acid=1:2) was flowed through the column at a fixedspeed (4 ml/h) for continuous reaction, to obtain 400 g of reaction oilsor fats. The column temperature was 40-41° C. The unreacted caprylicacid and free fatty acids were removed from the obtained reaction oilsor fats by molecular distillation, and then subjected to dietary oils orfats purification steps (degumming, deoxidation, deodorization,decolorizing) obtain 8A8-containing oils or fats (triglycerides).

The 8A8 proportion of the obtained 8A8-containing oils or fats(triglycerides) was determined by gas chromatography andhigh-performance liquid chromatography to be 31.6 mole percent.(Incidentally, the proportions of 8P8, 8O8, 8L8, 8G8 and 8D8 were 0.6,7.9, 15.1, 5.2 and 4.8 mole percent, respectively. The fatty acids P, O,L, G and D bonded at the triglyceride 2-position represent palmiticacid, oleic acid, linoleic acid, γ-linolenic acid and dihomo-γ-linolenicacid, respectively, and therefore 8P8 represents1,3-capryloyl-2-palmitolein-glycerol, 8O8 represents1,3-capryloyl-2-oleoyl-glycerol, 8L8 represents1,3-capryloyl-2-linoleoyl-glycerol, 8G8 represents1,3-capryloyl-2-γ-linolenoyl-glycerol and 8D8 represents1,3-capryloyl-2-dihomo-γ-linolenoyl-glycerol). Separation andpurification from the obtained 8A8-containing oils or fats(triglycerides) by an established high-performance liquid chromatographymethod yielded 96 mole percent 8A8.

Example 3 Evaluation of Effect on Behavior Disorder of TGA40S byBehavioral Observation Test

The experimental groups consisted of 51 two- to three-month-old male ICRmice, divided into a control diet group (27 mice) and aTGA40S-containing diet group (24 mice), with the control diet orTGA40S-containing diet shown in Table 1 being given to each group. Eachgroup was further divided into non-restrained groups (non-restrainedcontrol diet group (9), non-restrained arachidonic acid (ARA) diet group(12)) and restrained groups (restrained control diet group (18),restrained ARA diet group (12)). The restraining was accomplished usinga wire mesh restraining tube, once for a 6 hour period three weeks afterthe start of feeding. The control diet or TGA40S-containing diet shownin Table 1 continued to be fed to each group for the remainingexperiment period. The TGA40S used for the TGA40S-containing diet wasthe product obtained in Example 1.

TABLE 1 Experimental diet Control diet TGA40S-added diet Casein (g/kg)200 200 DL-methionine 3 3 Corn starch 150 150 Sucrose 500 500 Cellulosepowder 50 50 Corn oil 50 45 Mineral AIN-76 35 35 Vitamin AIN-76 10 10Choline bitartrate 2 2 Vitamin E 0.05 0.05 TGA40S 0 5

Since the daily ingestion was approximately 5 g per mouse, the dailyintake of TGA40S was 25 mg per mouse. Also, since the total fatty acidsbonded to the arachidonic acid-containing oils or fats (triglycerides)prepared in Example 1 included 40 wt % arachidonic acid, the dailyintake of arachidonic acid was 10 mg per mouse.

The 6-hour restraint with a wire mesh restraining tube was immediatelyfollowed by a behavioral observation test. The behavioral observationtest was conducted by transferring the mouse into a new cage and thenafter 30 minutes observing and recording the behavioral indicesaccording to the Irwin neurobehavioral test method (sniffing, circling,rearing, jumping, digging). No difference was observed between thecontrol diet mice and ARA diet mice in the absence of restraint stress.However, the mice of the control diet group which had experiencedrestraint stress clearly exhibited increased rearing, indicatingcautious reaction, and reduced spontaneous movements such as sniffingand circling, compared to the non-restrained mice, whereas the micegiven TAG40S (arachidonic acid) recovered to the same level of behavioras the mice without restraint stress (FIGS. 1, 2).

Thus, for the first time it has been clearly demonstrated thatadministration of TGA40S improves behavioral patterns which have alteredas a result of stress, and that arachidonic acid exhibits an improvingeffect against behavioral disorders.

Example 4 Preparation of Capsules Comprising Arachidonic Acid-ContainingOils or Fats (Triglycerides)

Water was added to 100 parts by weight of gelatin and 35 parts by weightof food additive grade glycerin for dissolution at 50-60° C., to preparea gelatin coating with a viscosity of 2000 cp. Next, 0.05 wt % ofvitamin E oil was combined with the arachidonic acid-containing oils orfats (triglycerides) obtained in Example 1 to prepare filling 1. VitaminE oil was also combined at 0.05 wt % with oils or fats (triglycerides)containing 32 mole percent of the 8A8 obtained in Example 2 to preparefilling 2. Also, 50 wt % of the arachidonic acid-containing oils or fats(triglycerides) obtained in Example 1 was combined with 50 wt % fish oil(tuna oil the eicosapentaenoic acid and docosahexaenoic acid proportionsof the total fatty acids were 5.1% and 26.5%, respectively) and then0.05 wt % vitamin E oil was added to prepare filling 3.

Also, 80 wt % of arachidonic acid-containing oils or fats(triglycerides) was combined with 20 wt % fish oil (tuna oil: theeicosapentaenoic acid and docosahexaenoic acid proportions of the totalfatty acids were 5.1% and 26.5%, respectively) and then 0.05 wt %vitamin E oil was added to prepare filling 4. Separately, 0.05 wt % ofvitamin E oil was combined with the 99% arachidonic acid ethyl esterobtained in Example 1 to prepare filling 5. These fillings 1 to 5 wereused for production of soft capsules containing 180 mg of filling percapsule, obtained by capsule molding and drying by ordinary methods.

Example 5 Use for Oil Infusion

After combining 400 g of the oils or fats (triglycerides) containing 96mole percent 8A8 obtained in Example 2, 48 g of purified egg yolklecithin, 20 g of oleic acid, 100 g of glycerin and 40 ml of 0.1 Ncaustic soda and dispersing the mixture with a homogenizer, distilledwater for injection was added to make 4 liters. This was emulsified witha high-pressure spray emulsifier to prepare a lipid emulsion. The lipidemulsion was dispensed into plastic bags at 200 ml per bag and thensubjected to high-pressure steam sterilization treatment at 121° C. for20 minutes to prepare an oil infusion.

Example 6 Use for Juice

A 2 g portion of β-cyclodextrin was added to 20 ml of 20% aqueousethanol, and then 100 mg of the arachidonic acid-containingtriglycerides obtained in Example 1 (containing 0.05% vitamin E) wereadded thereto while stirring with a stirrer, and the mixture wasincubated for 2 hours at 50° C. After room temperature cooling(approximately 1 hour), stirring was continued while incubating for 10hours at 4° C. The resulting precipitate was recovered by centrifugalseparation and then washed with n-hexane and lyophilized to obtain 1.8 gof a cyclodextrin clathrate compound comprising arachidonicacid-containing triglycerides. A 1 g portion of this powder wasuniformly mixed into 10 L of juice to prepare a juice comprisingarachidonic acid-containing triglycerides.

1. A method for treating symptoms or diseases associated withstress-induced behavior disorders, which comprises administering aneffective amount of a composition to a patient in need thereof, whereinthe composition comprises arachidonic acid and/or a compound comprisingarachidonic acid as a constituent fatty acid, and wherein the symptomsor diseases associated with stress-induced behavior disorders are anadjustment disorder, an acute stress disorder, and/or a posttraumaticstress disorder.
 2. The method of claim 1, wherein said compoundcomprising arachidonic acid as a constituent fatty acid is anarachidonic acid alcohol ester, or a triglyceride, phospholipid orglycolipid wherein all or a portion of the constituent fatty acid isarachidonic acid.
 3. The method of claim 2, wherein the triglyceride inwhich all or a portion of the constituent fatty acid is arachidonic acidis a triglyceride having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position.
 4. Themethod of claim 3, wherein said medium chain fatty acids are selectedfrom among C6-12 fatty acids.
 5. The method of claim 1, wherein thecomposition comprises triglycerides which include a triglyceride inwhich all or a portion of the constituent fatty acid is arachidonicacid.
 6. The method of claim 5, wherein the arachidonic acid content ofsaid triglycerides which include a triglyceride in which all or aportion of the constituent fatty acid is arachidonic acid, is at least10wt % of the total fatty acids of the triglycerides.
 7. The method ofclaim 5, wherein said triglycerides which include a triglyceride inwhich all or a portion of the constituent fatty acid is arachidonicacid, are extracted from a microorganism belonging to the genusMortierella, Conidiobolus, Pythium, Phytophthora, Penicillium,Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora,Echinosporangium or Saprolegnia.
 8. The method of claim 5, wherein saidtriglycerides which include a triglyceride in which all or a portion ofthe constituent fatty acid is arachidonic acid, are triglyceridescontaining no eicosapentaenoic acid.
 9. The method of claim 1, whereinthe composition comprises triglycerides of which at least 5 mole percentconsists of a triglyceride having medium chain fatty acids bonded at the1,3-positions and arachidonic acid bonded at the 2-position.
 10. Themethod of claim 9, wherein said medium chain fatty acids are selectedfrom among C6-12 fatty acids.
 11. The method of claim 1, wherein saidcomposition is a food composition or pharmaceutical composition.
 12. Themethod of claim 11, wherein said food composition is a common food (foodand drink), functional food, nutritional supplement, food for specifiedhealth uses, preterm infant formula, term infant formula, infant food,maternal food or geriatric food.
 13. The method of claim 1, wherein thecomposition further comprises docosahexaenoic acid and/or a compoundcomprising docosahexaenoic acid as a constituent fatty acid.
 14. Themethod of claim 13, wherein said compound comprising docosahexaenoicacid as a constituent fatty acid is a docosahexaenoic acid alcoholester, or a triglyceride, phospholipid or glycolipid wherein all or aportion of the constituent fatty acid is docosahexaenoic acid.
 15. Themethod according to claim 13, wherein the composition is characterizedin that the arachidonic acid/docosahexaenoic acid ratio (by weight) inthe combination of said arachidonic acid and docosahexaenoic acid is inthe range of 0.1-15.
 16. The method of claim 1, wherein the compositiondoes not contain eicosapentaenoic acid, or the composition compriseseicosapentaenoic acid at an amount that does not exceed ⅕ of thearachidonic acid in the composition.
 17. The method of claim 5, whereinsaid composition is a food composition or pharmaceutical composition.18. The method of claim 9, wherein said composition is a foodcomposition or pharmaceutical composition.
 19. The method of claim 5,wherein the composition further comprises docosahexaenoic acid and/or acompound comprising docosahexaenoic acid as a constituent fatty acid.20. The method of claim 9, wherein the composition further comprisesdocosahexaenoic acid and/or a compound comprising docosahexaenoic acidas a constituent fatty acid.
 21. The method of claim 5, wherein thecomposition does not contain eicosapentaenoic acid, or the compositioncomprises eicosapentaenoic acid at an amount that does not exceed ⅕ ofthe arachidonic acid in the composition.
 22. The method of claim 9,wherein the composition does not contain eicosapentaenoic acid, or thecomposition comprises eicosapentaenoic acid at an amount that does notexceed ⅕ of the arachidonic acid in the composition.